Stacey Jose's CIAP Aptamer Project (2017)

Calf-Intestinal Alkaline Phosphatase-Aptamer Diagnostic for Polycystic Ovary Syndrome

Introduction and Background

One in ten adult women are affected by an imbalance of reproductive hormones and levels of androgens leading to the disorder polycystic ovary syndrome (PCOS), in which the ovaries are abnormally enlarged due to the collection of fluid in the follicles, creating cyst sacs (Office on Women’s Health, 2016) (Mayo Clinic, 2014). This endocrine imbalance is a result of insulin resistance and higher sensitivity of androgens, making PCOS the leading endocrine disorder among women, both pre- and post-menopausal (Carragher, 2015). Further effects such as irregular and prolonged menstrual cycles, weight gain, excess hair growth and acne characterize those with the syndrome, giving rise to complications such as high cholesterol levels, type II diabetes, high blood pressure, and infertility (Mayo Clinic, 2014). Many problems lie within diagnosing the syndrome, as up to 70% of women who have PCOS have been reported to be misdiagnosed or undiagnosed (Carragher, 2015). Additionally, the lack of treatment targeted to eradicate PCOS depends on preventative methods or symptom reducers, such as diet and exercise or oral birth control (Carragher, 2015).

The abnormal hair growth-caused PCOS has been tied with high levels of the adrenal steroid dehydroepiandrosterone sulfate (DHEA-SO4), utilized to monitor the progression of puberty in young women with hyperandrogenism (Siemens, n.d.). As there is no true treatment for the syndrome, the purpose of this project is to develop an aptamer diagnostic tool as the new specific diagnostic. With the help of a target protein, DHEA-SO4 can act as marker to be conjugated with the protein molecule for assessment within the body. Calf-intestinal alkaline phosphatase, commonly known as CIAP or CIP, is commonly conjugated with DHEA-SO4 for this purpose.

An aptamer is an oligonucleotide sequence with a high binding specificity for a specific target molecule, ranging from proteins to cells. Aptamers are now considered better alternatives to antibodies, as the former is more inexpensive for production, smaller for access, more stable in varying environments, more selective for targets, and made in vitro instead of in vivo and unable to illicit an immune response (BasePair, n.d.). The practical uses with aptamers include therapeutic applications to inhibit a protein’s function or specific pathway, diagnostic applications for signaling the aptamer’s analyte of interest, drug delivery applications for the coupling of the aptamer with a drug to target its delivery, and more. The aptamer being selected for in this process will have a diagnostic application; once the marker DHEA-SO4 is conjugated “in a human protein-based matrix” to CIAP, an aptamer suitable with a bodily environment can select against the target to monitor the development of PCOS in higher levels of the steroid (Siemens, n.d.).

In order to find such an aptamer, the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in vitro selection method is utilized, in which the N50 RNA pool is introduced to the biotinylated CIAP target for the bound species to be immobilized on the streptavidin beads due to the biotin-avidin affinity pair while the unbound sequences are removed through multiple washes. The bead based aptamer selection is performed in PBS selection buffer, with the binding and selection of sequences within body-suitable temperatures. Immobilization of the bound species with target on the beads, reverse transcription of the bound RNA into ssDNA, cycle course and large-scale polymerase chain reactions (PCR) for tested amplification of dsDNA, concentration of products with ethanol precipitation, transcription into RNA for visualization with a PAGE gel, and elution of the RNA band for further rounds of selection.

The CIAP target enzyme has a formula weight of 69 kDa or 69,000 g/mol and functions as a dimer with other protein subunits (Fosset, 1974). Alkaline phosphatase is found in many organisms, but this particular enzyme is naturally found in bovine mucous membranes in intestinal tissue (Fosset, 1974). There is no need for phosphatases or excess enzyme to reduce costs, and the target acts on unincorporated dNTPs for removal (NEB, n.d.). The enzyme CIAP has an active site to bind to calcium and zinc ions or nucleic acids as the target is an activator of Mg+2, Zn, and Ca+2, and it operates best in a basic pH, such as 9.8, but is inhibited at a pH below 4.5 (Yan, 2003). Its storage buffer consists of 10 mM Tris, 0.15 M NaCl, 0.1 mM ZnCl2, 1 mM MgCl2 and has a pH of 8.0 in 50% glycerol. Because the storage buffer has a pH too basic to function in the body, it is not used as a selection buffer; instead, a Phosphate Buffered Saline (PBS) buffer with a more neutral pH of 7.4 and the correct monovalent salts suitable for CIAP can be utilized. A RNA aptamer was found in the Aptamer lab at the University of Texas at Austin to select against CIAP to be used as an inexpensive alternative to antibodies for an ELISA test (Huynh et al., 2015).

An RNA aptamer selection kept at ideal conditions is underway to select against CIAP and has been immobilized with magnetic beads to reduce the variant RNA pool and introduced to the target for the reverse transcription of the bound series into DNA and amplification with polymerase chain reactions for elution of RNA. Preliminary results are currently in the first round of selection with an insufficient concentration of eluted RNA for the second round. After multiple rounds of selection, a potential aptamer against the CIAP target can be conjugated with DHEA-SO4 act as a diagnostic monitor in the body of increased levels of androgens causing PCOS. With the objectives of repeating an RNA aptamer selection against the alkaline phosphatase and coupling it with the steroid, the monitoring of the progression of PCOS can ease the process of determining treatment before further complications and reduce cases of misdiagnosis.

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References

[1] Alkaline Phosphatase, Calf Intestinal (CIP). New England BioLabs. N.p., n.d. Retrieved from https://www.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip.

[2] Aptamers vs. Antibodies. Base Pair Biotechnologies. N.p., n.d. Retrieved from https://www.basepairbio.com/aptamers-vs-antibodies/.

[3] Carragher, Mike. The Most Misdiagnosed and Mistreated Hormonal Problem in Women. The Body Well, 7 July 2015. Retreived from www.thebodywellusa.com/the-most-misdiagnosed-and-mistreated-hormonal-problem-in-women/.

[4] DHEA-SO₄. Siemens Immulite 2000. N.p., n.d. Retrieved from http://labmed.ucsf.edu/labmanual/db/resource/proc-DHEA-S_SeimensImmulite.pdf.ma

[5] Fosset, Michel, Danielle Chappelet-Tordo, and Michel Lazdunski. Intestinal alkaline phosphatase. Physical properties and quaternary structure. Biochemistry 13.9 (1974): 1783-1788. Retrieved from http://pubs.acs.org/doi/abs/10.1021/bi00706a001.

[6] Huynh, Vincent, et al. Alternative ELISA Using a RNA Aptamer against Calf Intestinal Alkaline Phosphatase. The FASEB Journal 29.1 Supplement (2015): 562-6. Retrieved from http://www.fasebj.org/content/29/1_Supplement/562.6.

[7] Kulbachinsky, A. V. REVIEW: Methods for Selection of Aptamers to Protein Targets. Biochemistry Moscow. N.p., 9 Feb. 2006. Retrieved from http://protein.bio.msu.ru/biokhimiya/contents/v72/full/72131505.html.

[8] Phosphatase, Alkaline. Phosphatase, Alkaline - Worthington Enzyme Manual. N.p., n.d. Retrieved from http://www.worthington-biochem.com/bap/default.html.

[9] Polycystic Ovary Syndrome (PCOS) (2014). Mayo Clinic. N.p. Retrieved from http://www.mayoclinic.org/diseases-conditions/pcos/home/ovc-20342146.

[10] Polycystic Ovary Syndrome (2016). Office on Women's Health. N.p. Retrieved from https://www.womenshealth.gov/a-z-topics/polycystic-ovary-syndrome.

[11] Yan, ShuLian, et al. Effect of extraneous zinc on calf intestinal alkaline phosphatase. Journal of protein chemistry 22.4 (2003): 371-375. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/13678301.